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HANDLING PLEURAL FLUID SAMPLES FOR ROUTINE …

Jos M. PorcelPleural Diseases Unit, Department of Internal Medicine, Arnau de Vilanova University Hospital, Bio-medical Research Institute of Lleida, Lleida, Spaine-mail: removal of a small volume of PLEURAL FLUID for analysis (diagnostic thoracentesis) is performed to identify the cause of an effusion. The proper collec-tion and HANDLING of FLUID specimens is of paramount importance as it may affect the reliability and accuracy of analyses. Inadequately trained clinicians sometimes send a single vacuum bottle or large syringe to be cir-culated through various laboratory sections or even different laboratories. Often, a sample may contain a large blood or fibrin clot as the result of inadequate anticoagulation or mixing. This article briefly describes the optimal conditions for the collection, transporta-tion, and processing of PLEURAL FLUID SAMPLES in rou-tine clinical practice, thus avoiding potential sources of preanalytical PLEURAL FluidPleural FLUID specimens are obtained with a needle and syringe under aseptic conditions.

hydrogenase [LDH], which may experience a reduction in concentrations from day 2 onward), or cytomorphol-ogy (12,13). Conversely, refrigeration inhibits the viabil-

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Transcription of HANDLING PLEURAL FLUID SAMPLES FOR ROUTINE …

1 Jos M. PorcelPleural Diseases Unit, Department of Internal Medicine, Arnau de Vilanova University Hospital, Bio-medical Research Institute of Lleida, Lleida, Spaine-mail: removal of a small volume of PLEURAL FLUID for analysis (diagnostic thoracentesis) is performed to identify the cause of an effusion. The proper collec-tion and HANDLING of FLUID specimens is of paramount importance as it may affect the reliability and accuracy of analyses. Inadequately trained clinicians sometimes send a single vacuum bottle or large syringe to be cir-culated through various laboratory sections or even different laboratories. Often, a sample may contain a large blood or fibrin clot as the result of inadequate anticoagulation or mixing. This article briefly describes the optimal conditions for the collection, transporta-tion, and processing of PLEURAL FLUID SAMPLES in rou-tine clinical practice, thus avoiding potential sources of preanalytical PLEURAL FluidPleural FLUID specimens are obtained with a needle and syringe under aseptic conditions.

2 The syringe and needle used to inject the local anaesthetic ( , 2% mepivacaine or lidocaine) into the epidermis and pa-rietal pleura should not be used to obtain the PLEURAL FLUID sample (1). Other than the issue of dilution, the acidic local anaesthetic might artificially lower the pH of the aspirated PLEURAL FLUID (2). In small parapneu-monic effusions, this could lead to a failed prediction as to whether a chest tube will be necessary (2).Specimen CollectionBetween 20 to 40 mL of PLEURAL FLUID is needed for a complete analysis, which includes biochemical, cyto- HANDLING PLEURAL FLUID SAMPLES FOR ROUTINE ANALYSESRUT N ANAL ZLER N PLEVRAL SIVI RNEKLER N N KULLANIMIA bstractDiagnostic thoracentesis requires approximately 20 to 40 mL of PLEURAL FLUID . The FLUID should be placed in EDTA- or heparin-treated tubes for biochemical, microbiological, and cytological analyses when appropriate. The addi-tional use of blood culture bottles improves bacterial isolation.

3 Ideally, FLUID specimens should be sent immedi-ately to the laboratory and processed within 2 hours. If a delay is expected, the sample should be maintained at 4 C until analysis, except for microbiological cultures. A delayed analysis of more than 48 hours is unacceptable, although the cytomorphological features of refrigerated SAMPLES are well preserved for at least 72 words: PLEURAL FLUID , PLEURAL effusion, specimen han-dling zetTan sal torasentez yakla k 20 ila 40 mL plevral s v gerek-tirir. Uygun oldu unda s v ; biyokimyasal, mikrobiyolojik ve sitolojik analizler i in EDTA -veya heparin- ile muamele edilmi t plere konmal d r. Ek olarak kan k lt r i elerinin kullan m bakteriyel izolasyonu art r r. deal olarak, s v rnekleri laboratuvara hemen g nderilmelidir ve 2 saat i inde i leme al nmal d r. Bir gecikme bekleniyorsa rnek, mikrobiyolojik k lt r haricinde, analize kadar 4 C de muha-faza edilmelidir. Buzdolab ndaki rneklerin sitomorfolojik zellikleri en az 72 saat i in iyi korunuyor olmakla beraber 48 saatten daha fazla gecikmi analiz kabul kelimeler: Plevral s v , plevral ef zyon, rnek kulla-n m Derleme19logical and, when an infection is suspected, microbio-logical studies (3).

4 Specimens should be collected in tubes containing anticoagulants, such as ethylenedi-aminetetraacetic acid (EDTA; lavender top) or heparin (green top), in order to avoid clotting and cell clump-ing, which may give inaccurate cell counts and differ-entials. In one study, leukocyte cell counts from plain tubes ( , without anticoagulant) were significantly lower (by about 50%) than those obtained from EDTA-treated tubes (4).The recommended distribution of PLEURAL FLUID for various analyses is outlined in Table 1. One 5 mL tube is enough for biochemical analysis. For pH measurements, it has traditionally been taught that FLUID should be col-lected anaerobically in a heparinised syringe and sub-mitted to the laboratory on ice (5). More commonly, the FLUID sample is obtained using an unheparinised syringe and then transferred to a heparinised tube. This ma-noeuvre does not cause a clinically relevant increase in pH, provided care is taken to avoid significant exposure to air (6).

5 Nor is it necessary to pack the FLUID sample in ice, as long as the pH determination is performed within 4 hours following extraction (7,8). Ideally, the measure-ment should be obtained using a blood gas machine. In grossly purulent specimens, the measurement of pH or any assay, other than a Gram stain and culture, is of no value and should not be conducted as the clinical decision is straightforward; that is, chest drainage is re-quired for empyemas regardless of PLEURAL pH value (3). Plus, pus may clog and damage the analyser. Factors influencing PLEURAL FLUID pH have less effect on glucose concentration, suggesting that the latter can be used as an acceptable alternative to the former (8).If microbiological analysis is indicated, inoculation of 2 to 5 mL PLEURAL FLUID into aerobic and anaerobic blood culture bottles, in addition to sending SAMPLES in a sterile tube for Gram stain and standard processing, increases the sensitivity of bacterial cultures by 20% (9).

6 For PLEURAL tuberculosis, the inoculation of 5 mL into liquid culture media is as beneficial (50% positiv-ity) as the use of larger volumes (10).While the volume of PLEURAL FLUID necessary for the maximum yield of cytological analysis is controversial, some guidelines suggest submitting at least 20 mL (11). The larger the sediment to be obtained in order to maximise the yield of cell blocks, the greater the volume of FLUID that needs to be FLUID Transportation and ProcessingFresh FLUID should be promptly transported to the laboratory at ambient temperature. The maximum ac-ceptable time delay before the processing of PLEURAL FLUID specimens in the laboratory is 2 hours (5). If a lon-ger delay is expected, the specimen should be stored in the refrigerator at 4 C, except for microbiological cultures. Refrigerated storage for up to 48 hours has no significant effect on the total leukocyte count or dif-ferential, biochemical parameters (except lactate de-20 HANDLING PLEURAL FLUID SAMPLES FOR ROUTINE ANALYSEST able 1.)

7 ROUTINE tests for the evaluation of PLEURAL effusionsTest Submission method Volume, mL CommentsCell count and Heparin- or EDTA-treated tube 3-5* Modern automated analysers are as reliable asdifferential microscopic analysispH Heparinised syringe ( , arterial 2* Admixture of sample with local anaesthetic, blood gas syringe) or tube exposure to air, use of certain analysers, or Optional transportation on ice delayed analysis may cause significant alterations in the pH valueBiochemistries Heparin- or EDTA-treated tube, 3-5* ROUTINE FLUID chemistries include protein, lactate or tube without additives dehydrogenase, glucose and, if available, adenosine deaminaseCytological profile Heparin-, EDTA-, or citrate-treated 10-20 Yield increases if cell blocks and tube or container immunocytochemistry, in addition to smears, are evaluatedGram stain and Heparin- or EDTA-treated tube, along 5-10 Nucleic acid amplification tests for differentbacterial cultures with blood culture bottles microorganisms, as well as pneumococcal antigen tests, can be performedMycobacterial Heparin- or EDTA-treated tube 5 Liquid media are more sensitive thancultures conventional solid media* A total volume of 3 5 mL is enough for the sequential measurement of pH, cell counts.

8 And biochemistrieshydrogenase [LDH], which may experience a reduction in concentrations from day 2 onward), or cytomorphol-ogy (12,13). Conversely, refrigeration inhibits the viabil-ity of certain microorganisms and, therefore, should be discouraged for specimens sent for microbial studies. In any case, delays of over 48 hours are multichannel analysers are used to as-sess PLEURAL biochemical parameters. Firstly, the an-ticoagulant-coated tube containing the FLUID sample is used for measuring both pH in a blood gas analy-ser, and total (red blood cells and leukocytes) and differential (polymorphonuclear vs. mononuclear leu-kocytes) cell counts in an automated cell counter (or less frequently, manually) after gentle agitation. Then, the tube is centrifuged at 3000 rpm for 15 minutes at room temperature, and the supernatant is tested for protein, LDH, glucose, and adenosine deaminase. Op-tional PLEURAL FLUID biochemistries include albumin and cholesterol (suspicion of misclassified transudate), amylase (pancreatitis or oesophageal rupture), triglyc-erides (chylothorax), C-reactive protein (bacterial in-fection), and tumour markers (malignancy) (3).

9 In the microbiology laboratory, Gram stain and both aerobic and anaerobic cultures should be set up. After high-speed centrifugation of the specimen ( , 3000 rpm x 15 minutes), the sediment is used to prepare the smears (Gram stain and acid fast stain for Mycobac-terium spp.) and inoculate the culture media. Usually, blood agar, chocolate agar, MacConkey agar and, if the sample has been properly obtained, fastidious an-aerobic agar plates are prepared, incubated at 35-37 C and monitored for microbial growth for a minimum of 4 days. Alternatively, blood culture bottles containing PLEURAL FLUID are incubated and, if positive, subcultures are then performed on the appropriate solid media to identify the microorganism (9). For mycobacterial iden-tification, the use of liquid culture-based techniques, such as BACTEC and microscopic-observation drug-susceptibility (MODS) systems, provides higher yields and faster results than solid conventional media (Low-enstein-Jensen) (14).

10 As far as cytological examinations are concerned, it has been demonstrated that cellular integrity is well preserved for up to 72 h with appropriate refrigeration (2-8 C) (13). The ROUTINE cytopathological examination of PLEURAL FLUID involves the preparation of both cytospin smears and cell blocks. There are various fixation and staining techniques for cytospin smears and cell blocks prepared from the sediment after centrifugation. Slides are usually fixed in 95% isopropyl alcohol and stained with the Papanicolaou method, which clearly brings out nuclear details, thus allowing better identification of malignant cells (15). Moreover, cell blocks fixed in formalin are stained with haematoxylin and eosin, and represent the ideal specimens for immunocytochemis-try. A little-known fact is that cytospin material, includ-ing previously Pap-stained slides, can also be used for immunocytochemical analysis. In this author s experi-ence, limited immunocytochemical panels that include epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA), calretinin, and thyroid transcription fac-tor-1 (TTF-1) are especially useful for the differential diagnosis between reactive mesothelial cells, adeno-carcinoma, and mesothelioma (16).


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